Selenium-containing nucleosides are natural components of several tRNA species in Methanococcus vannielii. In the present study, the incorporation of selenium into these macromolecules was investigated using sonic extracts of M. vannielii. Nucleoside analysis of the Selenium 75-labeled tRNAs from these in vitro reaction mixtures demonstrated that the selenium was present in Selenium 75-labeled nucleosides identical to the two naturally occurring 2-selenouridines produced in vivo. Incorporation of selenium into these nucleosides was ATP-dependent and was maximal after twenty minutes. Addition of O-acetylserine enhanced the activity two- to three-fold, implicating a role for selenocysteine in the reaction. Added L-selenocysteine could function as a selenium donor, but the D isomer and D,L-selenomethionine were inactive. RPC-5 chromatography of bulk tRNA isolated from M. vannielii grown on radioactive selenite separated five major species of seleno-tRNAs. The amino acid-accepting activity of these tRNAs was investigated.